Carrier Rates in the Midwestern United States for GJB2 Mutations Causing Inherited DeafnessFREE

Author Affiliations: Molecular Otolaryngology Research Laboratories, Department of Otolaryngology, Head and Neck Surgery (Drs Green and Smith and Mr McDonald), Department of Pediatrics (Mr Scott and Dr Sheffield), and the Howard Hughes Medical Institute (Mr Scott and Dr Sheffield), University of Iowa Hospitals and Clinics, and the Department of Statistics and Actuarial Sciences (Dr Woodworth), University of Iowa, Iowa City. Drs Green, Sheffield, and Smith and Mr Scott contributed equally to this work.

ABSTRACT

Context Mutations in the GJB2 gene are the most
common known cause of inherited congenital severe-to-profound deafness.
The carrier frequency of these mutations is not known.

Objectives To determine the carrier rate of deafness-causing
mutations in GJB2 in the midwestern United States and the
prevalence of these mutations in persons with congenital sensorineural
hearing loss ranging in severity from moderate to profound, and to
derive revised data for counseling purposes.

Design Laboratory analysis, performed in 1998, of samples from
probands with hearing loss for mutations in GJB2 using an
allele-specific polymerase chain reaction assay, single-strand
conformation polymorphism analysis, and direct sequencing.

Setting and Subjects Fifty-two subjects younger than 19 years
sequentially referred to a midwestern tertiary referral center for
hearing loss or cochlear implantation, with moderate-to-profound
congenital hearing loss of unknown cause, parental nonconsanguinity,
and nonsyndromic deafness with hearing loss limited to a single
generation; 560 control neonates were screened for the 35delG mutation.

Main Outcome Measure Prevalence of mutations in the GJB2
gene by congenital deafness status.

Results Of 52 sequential probands referred for congenital
sensorineural hearing loss, 22 (42%) were found to have GJB2
mutations. The 35delG mutation was identified in 29 of the 41 mutant
alleles. Of probands' sibs, all homozygotes and compound heterozygotes
had deafness. Fourteen of 560 controls were 35delG heterozygotes, for a
carrier rate expressed as a mean (SE) of 2.5% (0.66%). The carrier
rate for all recessive deafness-causing GJB2 mutations was
determined to be 3.01% (probable range, 2.54%-3.56%). Calculated
sensitivity and specificity for a screening test based on 35delG
mutation alone were 96.9% and 97.4%, respectively, and observed
values were 94% and 97%, respectively.

Conclusions Our data suggest that mutations in GJB2 are
the leading cause of moderate-to-profound congenital inherited deafness
in the midwestern United States. Screening of the GJB2
mutation can be offered to individuals with congenital deafness with
high sensitivity and specificity by screening only for the 35delG
mutation. A positive finding should establish an etiologic diagnosis
and affect genetic counseling.

Congenital severe-to-profound deafness affects 0.05% to 0.1% of children in the United States. In
most of these families, there is no history of hearing loss, and a
definitive etiology is only rarely established in the deaf child. The
majority of cases are ascribed to unknown or presumed genetic factors.
Estimates of the proportion of deafness due to genetic factors vary
from 20% to 76%.1- 4

Recent advances in the molecular genetics of deafness have
improved our ability to identify heritable hearing losses. Most
familial moderate-to-profound congenital deafness is inherited as an
autosomal recessive trait.5 Heterogeneity is high and, to date, 25 nonsyndromic recessive loci have been identified and numbered
sequentially DFNB1 through DFNB25 (DFN, deafness; B,
recessive; integer, order of discovery).5,6 Four relevant
genes also have been cloned: DFNB1/GJB2,7DFNB2/MYO7A,8,9DFNB3/MYO15,10 and
DFNB4/PDS.11

Interestingly, and unexpectedly, mutations in the GJB2 gene
have been found to be responsible for half of the severe-to-profound
autosomal recessive nonsyndromic hearing loss in
multiplex (multiple cases) families from France,
Britain, Tunisia, and New Zealand. This finding makes
DFNB1-related deafness the most common type of hereditary
congenital hearing loss in those countries.12 The
GJB2 gene encodes the connexin 26 (Cx26) component of gap
junctions, and in approximately two thirds of Cx26 alleles from persons
with DFNB1-related deafness, a single mutation is
found.13- 16 This mutation, the deletion of 1 of 6
contiguous deoxyguanosines, is referred to as either 35delG (preferred
nomenclature as recommended by the Nomenclature Working
Group17) or 30delG.

In this article, we report the carrier frequency of the 35delG mutation
in the midwestern United States and estimate the prevalence of
GJB2 mutations in persons with congenital sensorineural
hearing loss. These data are used to assess the impact of GJB2
as a cause of autosomal recessive nonsyndromic hearing loss.

METHODS

Subject Accrual

Subjects were younger than 19 years and had congenital sensorineural
hearing loss. They were sequentially accrued from hearing loss and
cochlear implant referrals to an otolaryngology clinic in the
midwestern United States. Excluded from this study were persons from
consanguineous populations and those with syndromic, mild, unilateral,
acquired, or dominant types of hearing loss. Less than 20% of the
referral population was excluded. Evaluation included a complete
history taking and physical examination, audiometry, and, in 90% of
cases, a temporal bone computed tomographic scan. Hearing loss ranged
from moderate to severe at some frequencies (33%), but was most often
severe to profound at all frequencies (67%).

The control population consisted of 560 randomly selected neonates born
in the midwestern United States in 1995.18 As part of the Iowa Neonatal Screening Program, blood was obtained and placed on
filter-paper cards for each child born in Iowa. These cards were
rendered anonymous, assigned a random number, and used for DNA
extraction. We used only those DNA samples that had served successfully
as an amplification template in an unrelated project (representing
76.5% of neonatal cards).18 The population demographics of
the control sample reflect those of the state of Iowa: 94.8%
non-Hispanic white, 1.9% black, 1.7% Hispanic, 1.3% Asian and
Pacific Islander, and 0.3% American Indian.19 The white
population is largely of northern European origin; approximately 0.2%
of the population is Ashkenazi Jewish. All procedures were approved by
the University of Iowa Human Subjects Committee.

Screening

All DNA samples were screened for the 35delG mutation using an
allele-specific polymerase chain reaction (ASPCR). The 35delG
homozygotes were diagnosed as having GJB2-related hearing loss
and no other studies were performed. The 35delG heterozygotes were
screened by single-strand conformational polymorphism (SSCP) analysis
and direct sequencing (if shifts were observed) to determine whether a
second mutation was present. If no 35delG alleles were identified by
ASPCR, the GJB2 coding sequence was screened by SSCP and
sequenced if SSCP shifts were observed. In all cases with only a single
coding sequence mutation, the noncoding exon of GJB2 (exon 1)
was sequenced.

The ASPCR was performed using 40 ng of human DNA in an 8.4-µL PCR
reaction containing 1.25 µL of PCR buffer (100 mmol of
tris-hydrochloride, pH 8.8, 500 mmol of potassium chloride, 15 mmol of
magnesium chloride, 0.01% wt/vol gelatin); 200 µmol each of dATP,
dCTP, dGTP, dTTP; 25 pmol each of either normal or mutant primer and
the common primer; and 15 pmol each of control primer A,
5‘-CCCACCTTCCCCTCTCTCCAGGCAAATGGG-3‘ and control primer B,
5‘-GGGCCTCAGTCCCAACATGGCTAAGAGGTG-3‘.20 For 35delG ASPCR
the normal, mutant, and common primers have been described
previously.21 For M34T, the primers were:
normal=5‘-GCTCACCGTCCTCTTCATTTTTCGCATTCT-3‘,
mutant=5‘-GCTCACCGTCCTCTTCATTTTTCGCATTTC-3‘, and
common=5‘-TAGGAGCGCT-GCGTGGACACGAAGATCAG-3‘. Samples
were denatured at 95°C for 5 minutes, followed by 32 cycles at 95°C
for 40 seconds, 60°C for 30 seconds, and 72°C for 1 minute. The PCR
products were analyzed by electrophoresis in a 1.5% agarose gel
containing ethidium bromide. The control primers amplified a 360–base
pair (bp) product, which served as an internal control. The 35delG and
M34T products were 202 bp and 199 bp in length, respectively. For each
ASPCR run, sequence-tested 35delG-positive and 35delG-negative DNA
samples were examined as controls. Sequencing of 14 positive and 9
negative samples demonstrated complete concurrence between ASPCR
results and DNA sequencing; this number of samples was considered to be
adequate confirmation of ASPCR sensitivity.

The SSCP was carried out in 3 reactions using primer pairs
Cx26-1/Cx26-3, Cx26-10/Cx26-15, and Cx26-5/Cx26-6, as noted
earlier.21 Amplification of exon 1 was performed with
primers 5‘-TCTTTTCCAGAGCAAACCGC-3‘ and 5‘-GGGCAATGCGTTAAACTGGC-3‘ using
Advantage GC Genomic Polymerase Kit (Palo Alto, Calif) according to the
manufacturer's instructions. The PCR products were cleaned using a
QIAquick PCR purification kit (Qiagen, Chatsworth, Calif) and directly
sequenced. Control samples were screened for 35delG by ASPCR.

Statistical Analysis

Statistical comparison of results was performed using the 1-sided
Fisher exact test. Since it is only meaningful to determine whether a
mutation is statistically more common in the deaf population in
comparison with the hearing population, the 1-sided, rather than the
2-sided, P value was determined in power analysis (described
below). To determine whether there were
any statistically significant differences in the
35delG carrier rates between studies, the Fisher exact test was used
due to the small sample sizes of the other studies (discussed below).
There are a number of conflicting theories on how to calculate the
2-tailed P value of a Fisher exact test; of these, an accepted
theory is to double the 1-sided P value. Since none of our
comparisons was statistically significant in a 1-tailed comparison, and
since all 2-tailed P values are greater than the corresponding
1-tailed P values, 2-tailed values are not given.

The power was computed via Monte Carlo simulations. We generated
replications of the data as binomial random variables, C for binomial
(128, 0.0234) corresponding to the control sample and D for binomial
(52, 0.117) corresponding to the deaf sample, with the assumption of a
5-fold increase in the control prevalence rate. For each simulation, we
computed the 1-sided tail area of the Fisher exact test and calculated
the power as the percentage of 1-sided rejections in 50,000
replications.

The quantities q and r, sensitivity, and specificity
(defined below) are functions of 3 binomial random variables. We
computed the probable error (median error) of sensitivity, specificity,
and the quantities of 2r
(q+r)2, 2
(q+r), and
q2+2qr (defined below) by
generating 10,000 replications of the 3 underlying binomial
random variables. For each replication, we calculated the values of the
dependent variables, producing 10,000 replications of each.
Positive and negative probable errors then were calculated from the set
of replications as the deviations of the first and third quartiles of
each function from its median and used to determine the upper and lower
limits of the probable range. Probable error for ROther and ROverall (defined below) were computed by the same method.
The variance of an estimated proportion (p) from a sample
population (n) is p(1−p)/n. The SE
is the square root of this variance.

RESULTS

Screening of 52 sequential probands referred for congenital
sensorineural hearing loss identified 22 individuals with GJB2
mutations (19 with mutations of both alleles; the individual with the
M34T/H100Y genotype was not considered to have GJB2-related
deafness [as described below]). The 35delG mutation was found in 29
of the 41 mutant alleles; 2 mutations, M34T and 167delT, were
identified twice; 8 other mutations occurred once (Table 1).

Table 2 presents
GJB2-related deafness prevalence in several subpopulations
with deafness. Mutations of GJB2 were present on both alleles
in 7 of 12 deaf sibships we studied. Examination of the sibs of
probands revealed that all homozygotes and compound heterozygotes had
hearing loss. The degree of loss could not be predicted by the
GJB2 genotype, as demonstrated in 1 family in which one child
had moderate and the other child profound hearing loss. Whether some
genotypes are associated with profound hearing loss more consistently
than other genotypes is not known.

Three persons carried only a single GJB2 mutation (genotypes
35delG/+, M34T/+, and R98Q/+). A
second mutation could not be demonstrated by sequencing both exons in
these individuals. The 35delG carrier was found to have dilated
vestibular aqueducts, a feature seen in approximately 10% of the
referral population but in none of the other GJB2 mutation
carriers. These 3 persons were not considered to have
GJB2-related deafness. The other person carrying GJB2
mutations who did not have GJB2-related deafness was the (Table 1).

Fourteen 35delG heterozygotes were identified in the
control population among 560 individuals, for a 35delG carrier rate of
2.5%±0.66% (SE). The M34T polymorphism was
identified in 3 (2.3%) of 128 unrelated Centre d'Etude du
Polymorphisme Humain controls (a standard reference group composed
primarily of persons of northern European ancestry22),
which is not significantly different from the prevalence in the deaf
population (2/52; P=.85, 1-sided Fisher
exact test). Despite the comparable prevalence of the M34T and 35delG
mutations in the general population, no M34T homozygotes or M34T/35delG
compound heterozygotes were identified. A 5-fold difference in M34T
carrier rates between the deaf and control population at
P=.05 can be
detected with the sample sizes of our
populations with a power of 0.68. Combining our data with similar data
(derived from individuals in the midwestern United States with
moderate-to-profound deafness [W. J. Kimberling, oral communication,
May 20, 1999]) obtained by Kelley et al16 increases the power to 0.96 with Monte Carlo error of about 0.5%. Thus, the M34T
mutation was not considered to cause deafness.

These data were used to estimate the proportion of the population
in the midwestern United States with moderate-to-profound congenital
GJB2-related deafness. Based on the Hardy-Weinberg law, this
proportion, NGJB2, can be represented as
(q+r)2=q2+2qr+r2,
in which q equals the chromosomal rate of the 35delG mutation
and r equals the chromosomal rate of all other GJB2
mutations that cause hearing loss in the compound heterozygote.

We determined the 35delG chromosomal rate experimentally to be 14 of
1120, and although r cannot be calculated similarly since all
other deafness-causing alleles of GJB2 are not known, it can
be estimated. If NOther represents the proportion of the
population in the midwestern United States with deafness due to other
causes (GJB2-unrelated deafness), then the subportion of these
individuals who are, coincidentally, 35delG carriers is approximately
2q × NOther. Based on these
definitions, the ratio of 35delG homozygotes to 35delG heterozygotes
among deaf individuals, h, can be represented as
q2/2qr+2q(NOther). Since the ratio of the proportions, NGJB2 and
NOther, equals the ratio of individuals with
GJB2-related deafness to individuals with
GJB2-unrelated deafness (18 and 34, respectively, in our
population), NGJB2/NOther = 18:34.

By substitution, h can be expressed as
q2/2qr+2q
(34:18)(NGJB2). When data reported by Kelley et
al16 are combined with
ours, h is 25:12 (Table 3). Therefore, a ratio of
25:12=q2/2qr+2q
(34:18)(q+r)2. Solving
this equation with q=14:1120 gives a value for
r of 0.00257. This value makes the carrier rate for
non-35delG GJB2 recessive deafness-causing mutations
approximately 0.51% (probable range, 0.38%-0.68%).

The carrier rate for all GJB2 recessive deafness-causing
mutations is the sum of the carrier rates for the 35delG mutation and
all other deafness-causing mutations, or 3.01% (probable range,
2.54%-3.56%). Based on values for q and r, the
proportion of the population with GJB2-related congenital
deafness, (q+r)2, is 22.7
(probable range, 15.1-31.9) per 100,000; the proportion with
congenital deafness due to other causes, but who meet the criteria for
this study, is 42.9 per 100,000. The subportion of the
population with GJB2-related congenital deafness and the
35delG mutation (q2+2qr)
is 22.0 (probable range, 14.7-30.7) per 100,000 (35delG
noncarrier is 0.66); and the subportion of the population with
GJB2-unrelated deafness coincidentally carrying the 35delG
mutation (2q [34:18]
[q+r]2) is 1.1 per
100,000 (noncarrier is 41.8).

Despite the degree of variability in the absolute values for each of
the subpopulations described above, the comparative ratios of
these subpopulations vary minimally, allowing calculation of the
sensitivity and specificity for detection of GJB2-related
hearing loss based on the presence of 35delG. The sensitivity of this
test is the true-positive prevalence (subportion of the population with
GJB2-related congenital deafness and the 35delG mutation)
divided by the population with GJB2-related congenital
deafness (22.0/22.7=96.9%, probable range,
95.4%-98.0%). The specificity of this test is the true-negative
prevalence (the subportion of the population with
GJB2-unrelated deafness not coincidentally carrying the 35delG
mutation) divided by the population with GJB2-unrelated
deafness (41.8/42.9=97.4%,
probable range, 97.0%-98.0%), which also
approaches 1−2q/1 since q is small.
The observed sensitivity (17/18=94%) and specificity
(33/34=97%) are comparable with these calculated
values. Inclusion of data from international sources12- 15
does not substantially alter these results.

These calculations assume the population is randomly mating with
respect to GJB2. The existence of population substructure,
particularly endogamous subpopulations, results in a decreased
proportion of heterozygotes (Wahlund effect) and an undercalculation of
r with an overestimation of test sensitivity for the
population as a whole. Other assumptions made in these calculations
include complete penetrance and lack of ascertainment bias (ie, equal
referral rates regardless of genotype), and negligible heterozygote
selection advantage, spontaneous mutation rate, and migration effects.
Deviation of the actual population from Hardy-Weinberg equilibrium due
to these factors is likely to be minimal and does not affect the order
of magnitude of the figures obtained with the possible exception of
assortative mating among the deaf, which is discussed below.

Bayesian analysis using these data permits delineation of the
recurrence risk for heritable deafness. The overall chance of having a
second deaf child (ROverall) is dependent on the proportion
of that population with GJB2-related hearing loss
(NGJB2), the proportion of that population with
GJB2-unrelated hearing loss (NOther), and the
individual recurrence risks for GJB2-related deafness
(RGJB2) and GJB2-unrelated deafness
(ROther). Assuming near-complete penetrance,
RGJB2 is approximately 25%. Therefore,
ROverall=NGJB2 × RGJB2+NOther × ROther =NGJB2 × 25%+NOther
× ROther.

This study has determined the proportions of sequential deaf probands
with GJB2-related
(NGJB2=18/52) and
GJB2-unrelated deafness
(NOther=34/52). The ratio, S, of
GJB2-related to GJB2-unrelated deafness among deaf
sibships is dependent on the relative fecundities, proband frequencies,
and recurrence chances of couples with a deaf child. Assuming no
differences in fecundity,
S=NGJB2 × RGJB2/NOther × ROther. Since S=41/42 (Table 2), and
NGJB2, NOther, and
RGJB2 are known, ROther can be shown
to be 13.6% (probable range, 10.6%-17.2%). The a priori chance,
ROverall, is then 17.5% (probable range,
15.0%-20.4%).

For a hearing couple, assortative mating among the deaf does not affect
ROverall, which is calculated exclusively from values
determined for hearing couples. For a deaf couple, recurrence chances
must be calculated independently and may vary from 0% to 100%,
depending on the etiologies of deafness and carrier statuses for
deafness-causing mutations. (Assortative mating results in increased
carrier rates of deafness genes among deaf persons who have multiple
deaf progenitors in comparison with deaf persons who do not have deaf
progenitors [ie, a deaf man with congenitally deaf parents and
grandparents is more likely to carry deafness genes], including genes
unrelated to the etiology of his own deafness.) Additionally, the
recurrence chance for a couple in which 1 spouse is deaf or has
nonpenetrant deafness is higher. Since the carrier rate of 35delG
(2.5%) is much greater than the rate of congenital hereditary deafness
from assortative couplings (>0.1% of the general population), the
calculation of carrier rates is not affected substantively by
assortative mating.

COMMENT

Mutations in GJB2 are the most common cause of
moderate-to-profound congenital inherited deafness in the midwestern
United States. This deafness etiology (requiring deafness-causing
mutations of both alleles) was found in 18 (35%) of 52 probands
evaluated for congenital, moderate-to-profound sensorineural hearing
loss (Table 1) and in more than 50% of multiplex sibships. Although
numerous deafness-causing mutations of this gene occur, a single
mutation, 35delG, predominates. Our data are consistent with other
national and international data in showing that 60% of persons with
GJB2-related deafness are homozygous for the 35delG
allele.13,14,16 The carrier rate as a mean (SE) in the
general population for this allele is 2.5% (0.66%) (Table 3). The
total carrier rate for all GJB2 deafness-causing mutations is
3.01% (probable range, 2.54%-3.56%). The corresponding predicted
prevalence of GJB2-related congenital hearing loss is 22.7
(probable range, 15.1-31.9) per 100,000 births. More than two
thirds of these individuals have profound or severe-to-profound hearing
loss, although there can be phenotypic variability in the degree of
loss even within the same family. The lower 35delG carrier rates noted
by Scott et al21 (1/100; P=.31)
and Kelley et al16 (2/96; P=.58)
(P values represent comparison with our results) are
consistent with stochastic variances due to smaller sample sizes. The
study by Morell et al23 showed a carrier rate of 1 in 173
among white college students. Although the difference between this
result and our results does not achieve statistical significance
(P>.09), it is important to note that the predicted
prevalence of 35delG homozygosity corresponding to this carrier rate
would be 0.8:100,000, a value inconsistent with observed
data.1- 4,14- 16

Data used to provide information for genetic counseling and estimates
of recurrence risks for hearing couples with a deaf child are taken
from segregation analyses of demographic data from the 1800s and from
large family surveys.24 Analyses using these sources
provided estimates of the chance of recurrence under these conditions
at 9.8%.25 Using the results derived from the sequentially
accrued probands in our study, we estimate the chance for a
normal-hearing couple to have a second deaf child to be 17.5%
(probable range, 15.0%-20.4%) provided the first deaf child meets the
criteria for inclusion in this study. This value is comparable with a
population-based estimate from Canada of
16%±3%.2

Several factors have contributed to this change in recurrence-risk
estimates, including an improved ability to identify syndromic forms of
deafness characterized by variable expressivity
and a decrease in congenital-acquired hearing
loss most recently reflected in the effect of rubella immunization on
rubella-induced deafness.26 These factors, and an increase
in population mobility leading to a loss of segregating communities
(thereby decreasing contributions from rare genes), act to increase the
proportion of deafness due to GJB2 mutations both in the
hearing and Deaf communities.

The sensitivity of a genetic screen for GJB2-related deafness
can be enhanced by examination for mutations beyond 35delG that are
known to cause deafness. In particular, the 167delT mutation appears to
be relatively common among Ashkenazi Jews, with a measured prevalence
of 4%, despite being rare in the general population.23
Screening for 167delT to identify GJB2-related deafness should
be considered for populations containing a sizable Ashkenazi Jewish
subpopulation. After identification of a single mutation in
GJB2, more exhaustive methods, such as SSCP and sequencing,
are recommended for screening for the presence of a second mutation.

These data support the future use of a genetic test, such as ASPCR,
designed to identify the 35delG GJB2 mutation, as a valuable
complement to audiometric screens to identify neonates with heritable
congenital hearing impairment in nonendogamous white populations of the
Midwest and other ethnically similar populations. Use of this test may
facilitate earlier habilitation in a substantial percentage of deaf
infants and ultimately may provide parents with valuable prognostic and
therapeutic information.

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